MECHANISMS OF E HISTOLYTICA ADHERENCE AND CYTOLYSIS

肠道原虫溶组织内阿米巴致病 在世界范围内；寄生虫的坚持和细胞溶解事件在 侵袭性阿米巴病的发病机制。我们的目标是定义 阿米巴黏附和细胞溶解的生化和分子基础 申请开发保护性疫苗的活动 对抗侵袭性阿米巴病。寄生虫体外黏附，介导性 由170 Kdalton半乳糖/N-乙酰-D-氨基半乳糖(Gal/GalNAc)组成 可抑制的表面凝集素，启动靶细胞的细胞溶解。 佛波酯刺激阿米巴溶细胞活性需要 阿米巴磷脂酶A活性，内吞囊泡的酸性pH- 致死性增加靶细胞内游离钙离子((Ca++))。 候选疫苗包括半乳糖/半乳糖凝集素，细胞溶解 寄生虫蛋白和主要阿米巴抗原。具体目标 本方案的方法是：(1)体内测定 纯化的Gal/GalNAc凝集素在沙土鼠中的疫苗效果！模型 肠道定植和肝脓肿的关系；(2)描述 蛋白激酶C(PKC)介导的调节和刺激 测定pborbol酯诱导的PKC胞浆的杀伤活性 膜转位与蛋白底物磷酸化；(3) 为了鉴定纯化的Gal/GalNAc凝集素的细胞毒性， 包括凝集素的结合，对(Ca++)的影响，以及 (4)为了确定靶细胞死亡的机制， 包括Ca++DNA降解、磷脂酶、 蛋白水解酶和膜“孔”；5)阿米巴蛋白的特性 它们通过以下途径参与细胞溶解活动(细胞蛋白) 溶组织蛋白基因缺失克隆的诱变选育 细胞溶解活性(Cyt-)，Cyt-与Cyt+阿米巴比较 双向凝胶电泳法和免疫印迹法鉴定 Cyt-突变体中的Cyt+蛋白改变，部分氨基酸 这些蛋白质的测序为合成这些蛋白质提供了信息 应用寡核苷酸探针筛选双歧杆菌Cyt+cDNAs 基因组文库及细胞色素T蛋白功能的测定(S) 从基因组中克隆的全长DNA的序列分析 抗细胞抗体文库及其对阿米巴细胞溶解作用的影响 活性；(6)确定免疫原性和疫苗 细胞色素T蛋白的功效及其识别的主要阿米巴抗原 用纯化的B-B免疫沙土鼠免疫人免疫血清 半乳糖苷酶融合蛋白。这项研究将扩大我们的 了解溶组织埃希氏菌并为消灭该病做出贡献 作为人类疾病的一个原因。

The enteric protozoan Entamoeba histolytica causes disease worldwide; parasite adherence and cytolytic events are crucial in pathogenesis of invasive amebiasis. Our objective is to define the biochemical and molecular basis of amebic adherence and cytolytic activities with application for development of a vaccine protective against invasive amebiasis. Parasite in vitro adherence, mediated by a 170 Kdalton galactose/N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable surface lectin, initiates cytolysis of target cells. Amebic cytolytic activity is stimulated by phorbol esters requires amebic phospholipase A activity, an acid pH in endocytic vesicles- lethal increase in target cell free intracellular Ca++ ((CA++)). Candidates for a vaccine include the Gal/GalNAc lectin, cytolytic parasite proteins, and major amebic antigens. The specific aims and methods of this proposal are: (1) to determine the in vivo vaccine efficacy of purified Gal/GalNAc lectin in gerbil! models of intestinal colonization and liver abscess; (2) to describe protein kinase C (PKC) mediated regulation and stimulation of cytolytic activity by determining pborbol esterinduced PKC cytosol- membrane translocation and protein substrate phosphorylation; (3) to characterize the cytotoxicity of the purified Gal/GalNAc lectin, including lectin binding, effects on (Ca++), and uptake by the target cell; (4) to determine the mechanism of target cell death, including the role of Ca++ DNA degradation, phorpholipases, proteases, and membrane "pores"; 5) to characterize amebic proteins which participate in cytolytic activity (Cyt proteins) by mutagenesis and selection of histolytica clones deficient in cytolytic activity (Cyt-), comparison of Cyt- to Cyt+ amoebae by two-dimensional gel electrophoresis and immunoblotting to identify Cyt+ proteins altered in Cyt- mutants, partial amino acid sequencing of such proteins providing information for synthesis of oligonucleotide probes to screen E. bistolytica Cyt+ cDNA and genomic libraries, and determining function(s) of Cyt proteins by sequence analysis of full length DNA cloned from the genomic library and effects of anti-Cyt antibodies on amebic cytolytic activity; and (6) to determine the immunogenicity and vaccine efficacy of Cyt proteins and the major amebic antigens recognized by human immune sera by immunization of gerbils with purified B- galactosidase fusion proteins. This research will expand our knowledge of E. histolytica and contribute to eliminating it as a cause of human disease.